Restoration of liver function in gunn rats without immunosuppression using transplanted...

Restoration of Liver Function in Gunn Rats without Immunosuppression Using Transplanted

Microencapsulated Hepatocytes

VIVEK DIXIT,' RUTH DARVASI,*. ' MARIKA ARTHUR,' MARIA BREZINA,' KLAUS LEWIN' AND GARY GITNICK' Departments of 'Medicine and 'Pathology, UCLA School of Medicine, Los Angeles, California 90024-1 684

Microencapsulation of cells within synthetic semipermeable membranes is a novel technique that enables the transplantation of cell cultures without the need for immunosuppression. We have previously shown that transplanted isolated encapsulated hepatocytes can provide sufficient short-term metabolic support to improve the survival of animals with galactosamine-induced fulminant hepatic failure. Here we have demonstrated the feasibility of isolated encapsulated hepatocyte transplantation in providing long-term metabolic liver support in Gunn rats. Gunn rats have a congenital inability to conjugate bilirubin and thus exhibit lifelong hyperbilirubinemia. We studied the feasibility of isolated encapsulated hepatocyte transplantation in restoring this specific liver function. Free hepatocytes, isolated from male Wistar rats, were microencapsulated with collagen within a trilayered sodium alginate-poly-L-lysine-sodium alginate membrane using techniques developed in our laboratory. A total of 45 Gunn rats underwent intraperi- toned transplantation with free hepatocytes (5 x lo7), isolated encapsulated hepatocytes (5 x lo7), control (empty) microcapsules or no transplant (untreated controls). Serum bilirubin levels were monitored daily for 10 days after transplantation, and subsequent weekly samples were obtained for up to 1 mo. Microcap- sules were studied by light and electron microscopy 1 mo after transplantation. During the first week after transplantation, the mean maximum reduction in serum bilirubin levels for the isolated encapsulated hepatocytes, free hepatocytes and control microcapsule transplanted groups was 45.7%, 18.6% and 14.3%, respectively. For up to 1 mo thereafter the mean reduction in serum bilirubin levels in these respective groups was 34.896, 13.5% and 3.38. Light and electron microscopy of the isolated encapsulated hepatocytes revealed preservation of a number of hepatocytes containing numerous mitochondria, smooth and rough en-

Received November 13, 1989; accepted June 27, 1990. This work was supported in part by a grant from the United Liver

Association. Las Angeles, California. 'Currently at Hadassah Hospital (Mt. &opus). Department of Gastroenter-

ology, Hebrew University, Jerusalem, Israel. Address reprint requests to: Gary Gitnick. M.D.. UCLA School of Medicine,

Department of Medicine, 10833 Le Conte Avenue, Los Angeles. California 90024-1684.

31/1/24801

doplasmic reticulum, Golgi bodies and glycogen. We conclude that isolated encapsulated hepatocyte transplantation was effective in significantly lowering serum bilirubin levels in the Gunn rat. Transplan- tation of isolated encapsulated hepatocytes is a unique approach to the restoration of at least one liver function in congenital liver deficiency. (HEPATOLOCY 1990; 12: 1342-1349.)

Restoration of liver function may improve quality of life and enhance survival of patients with acute liver failure, chronic liver disease and congenital metabolic liver disease. Except for whole organ liver transplantation, there are few satisfactory treatments to improve survival and quality of life for these patients. Liver transplantation is costly and complex, requiring highly sophisticated technology and support teams. Furthermore, a growing need exists for a simple, cost- effective liver biosupport system (LBS) that could (a) sustain patients with acute liver failure, (b) prepare patients for liver transplantation when a donor liver is not readily available (i.e., bridge to transplantation), and (c) improve the survival and quality of life for patients for whom liver transplantation is not a therapeutic option.

To date, efforts toward the development of an LBS have evolved from technology developed for the treatment of kidney failure. Thus research has been directed mainly toward the removal of metabolic waste products. Detoxification systems such as charcoal hemoperfusion, plasmapheresis and high-porosity membrane hemodialysis have been reported to promote the recovery of consciousness in patients with deep hepatic coma (1-3). These approaches only address one aspect of the liver's complex metabolic function. The healthy liver carries out a wide range of essential synthetic, catabolic, storage and excretory functions. Thus, an artificial LBS must also be capable of assuming all these functions. Of the numerous approaches being developed, the transplantation of isolated healthy hepatocytes shows the greatest promise of being able to assume the full range of liver functions (4-10).

The transplantation of hepatocytes to supplement failing or deficient liver function is not a new concept ( 11 ). Recent developments in cell culture techniques

1342

have facilitated the usit' of' transplanted hepatocytes with very promising results. Demetriou and colleagues (5) demonstrated very elegant1.v tha t transplantation of microcarrier-attached hepatocytes could supplement deficient liver function in two animal models of congenital metabolic liver defects: hyperbilirubinemia and analbuminemia. Additionally. the same group also showed that transplanted microcarrier-attached hepatocytes could significantly increase the survival of rats undergoing 90% partial hepatectomy ( 12) . The t rans- planted hepatocytes provided sufficient metabolic support to enable enough regeneration of the remain- ing liver mass to ensure the hepatectomized animals' survival.

In these studies, isolated hepatocytes were transplanted into either athymic mutant rats or allogen- ic animals that were immunosuppressed with cyclo- sporin A to prevent rejection of t h e transplanted cells (5 , 12, 13). These important studies have demonstrated the feasibility of hepatocyte transplantation for congenital metabolic liver diseasc. and acute liver failure. However, without adequate i in m unosu ppression, com - plications caused by tissue wjection remain a problem. In an earlier study it was reported that hepatocytes from different strains of rats were not rejected after intraperitoneal transplantation in Gunn rats ( 10). In tha t study both free and encapsulated hepatocytes not only had similar effect in lowering serum bilirubin levels in Gunn rats b u t were also not rejectcld after intraperitoneal transplantation. Our studies. as well as those of other groups, do not seem to support this observation (14-17). It has been reported tha t hepatocytes express major histocompatibility complex class I but not class I1 antigens (18). Although the expression of the latter has been hypothesized in the mediation of allograft tissue immunogenicity ( 19 1, not all experimental data support this hypothesis. Various in riitro and in cliuc) studies have demonstrated that pure hepatocytes a re immunogenic despite being major histocompatibility complex class I1 negative (18-20). To circumvent the complications of immune rejection of t h e transplanted hepatocytes, we and others have developed techniques to microencap- sulate isolated hepatocytes within a synthetic semipermeable membrane composed of sodium alginate and polylysine (7-9). The semipermeable membrane is de- signed to allow molecules such as albumin and clotting factors to freely permeate but is impermeable to antibodies and cells (21 1. Thus, microencapsulation of liver cells within synthetic semipermeable membranes is an innovative technique that enables the transplantation of cell cultures without the need for immunosuppression. We and others have recently shown that transplanting isolated encapsulated hepatocytes I IEH) can significantly increase the survival of animals with galactosamine-induced fulminant hepatic failure (22, 23). As an extension of our ongoing research, we have tested the hypothesis that transplantation of IEH can restore a specific congenital enzyme deficiency in G u n n rats that exhibit lifelong hyperbilirubinemia because of a n inherited inability to conjugate bilirubin.

Furthermore, we have sought to determine whether transplanted I E H are subject to rejection in the absence of immunosuppression

MATERIALS AND METHODS All prot.ocols were approved by the Chancellor's Animal

Research Committee (ARC 1% University of California, Los Angeles, according to the NIH C;riide for the Care and [ J s c of Laboraton, Animals.

Hepatocytes were isolated from 125 to 150 gm adult male Wistar rats ((:harles River Breeding Laboratories. Wilmington, MA) by an rrt-situ modification of the portal vein collagenase (Type IV, 200 Uiml; Worthington Biochemical Corp.. Freehold, N J I perfusion technique described by Seglen (24 1. Freshly isolated hepatocytes were filtered through a 9.5 km sieve to remove cell clumps and connective tissue debris. The partially purified hepatocyte suspension was then differentially centrifuged at 4 'C to yield a homogeneous suspension of individual hepatocytes. Only those hepatocyte suspensions with a viabilitv

85'4 were used for transplantation. Hepatocyte yield rou- tinely varied between 1.2 and 3.0 k 10" cells. Sterile technique was used throughout the isolation procedure. Isolated hepatocytes were suspended in RPMI 1640 before microencapsu- lat ion.

Recipient Animals. Free or isolated encapsulated hepato- cvtes and control (empty) microcapsules were aseptically transplanted into 185 gm 2 7.2 gm homozygous Gunn rats. These rats have a congenital deficiency of bilirubin-uridine diphosphate glucuronyltransferase activity and are unable t o conjugate bilirubin in bile and therefore exhibit lifelong. nonhemolytic, unconjugated hyperbilirubinemia.

Mirroencapsulation. Microencapsulation of isolated hepatocytes was carried out at room temperature using aqueous buffers at physiological pH. This procedure results in a < 5 5 loss in hepatocyte viability 7 ) . Briefly. isolated hepatocytes were microencapsulated within a collagen matrix enveloped by an ultra-thin sodium alginate-poly-1.-lysine-sodium alginate (APLA) copolymer membrane. The technique is a modification of' the procedure initially described by Lim (25) and subse- quently by O'Shea, Goosen and Sun (26 ) . Isolated hepatocytes were suspended in a mixture of 2% (viscosity = 266 cps) sodium alginate (Kelco Gel LV. Kelco, San Diego, CA) and 1.7 rnniol/L bovine dermal collagen (Vitrogen 100. Collagen Corp.. Palo Alto, CA). The mixture was then extruded through a droplet-generating apparatus to form microdroplets of approximately 300 to 700 km diameter. The alginate microdroplets were reacted with 28 pmoliL poly 1.-lysine (Sigma Chemical (h., St. Louis, MO) for exactly 15 min to facilitate the formation of an outer skin of polylysine on the surface of the alginate microdroplet. The polylysine is covalently bonded to the alginate exposed on the surface of the microdroplet ( 2 7 ) . The polylysine-coated algmate microdroplets were further reacted with a 0.2% solution of sodium alginate to form covalent linkages between the polylysine and the alginate, thereby completing the formation of the APLA membrane of the microcapsule. The formed microcapsules were supported internally by a matrix of collagen and hepatocytes. These microcapsules are suhsequently referred to as IEH. Each milliliter of IEH contained approximately lo7 hepatocytes.

Transplantation Studies. Free hepatocytes (FHC), freshly isolated from male Wistar rats. were microencapsulated with collagen within a trilayered sodium APLA membrane by techniques developed in our laboratory. Five milliliters of IEH, a n equivalent quantity of FHC and control empty microcap-

Hepatocyte Isolation from Donor Animals.

1344 DIXIT ET AL. HEPATOLOGY

I

r I - Group I --b Group 2 - Group 3 -u- Group 4

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28

Time (days)

FIG. 1. Serum bilirubin levels in Gunn rats after transplantation of IEH (group l), FHC (group 2), control (empty) microcapsules (group 3) or no transplant (group 4). Group 1 animals show a highly significant (p < 0.001) decrease in serum bilirubin levels after IEH transplantation. Groups 2, 3 and 4 show no significant (p < 0.70) change.

TABLE 1. In uiuo studies: percentage decrease in bilirubin levels after transplantation of IEH, FHC and control (empty) microcapsules

Days after transplantation

Group 0 1 3 5 7 10 21 28

IEH 0 28.6 ? 4.5" 43.4 ? 4.0" 35.1 2 3.3" 37.0 2 4.4" 33.9 ? 5.8" 44.4 ? 6.8" 35.4 ? 4.6" FHC 0 11.3 ? 4.0 17.4 ? 3.0h 19.3 t 4.0" 12.7 4.0b 16.0 5 5.0b 20.4 ? 8.0 16.5 ? 9.0 Control 0 3.5 1.4 11.9 5 5.0 19.7 2 7.0 9.5 2 4.3 18.3 2 9.0 1.4 2 1.1 6.7 5 6.7

"p < 0.01 compared with FHC and control groups. 'NS = not significant compared with control group. Above values are mean ? S.E.M.

sules (containing no hepatocytes) were suspended in 10 ml of RPMI 1640 and were intraperitoneally transplanted into Gunn rats by sterile techniques. The FHC or microcapsule suspension was injected into the peritoneal cavity of lightly anesthetized (halothane) Gunn rats with a 12-gauge needle. The puncture in the abdomen was closed with a skin stapler. Serum bilirubin levels were measured in the blood samples obtained by cutting a 0.5 mm slice of the animal's tail. Bleeding was stopped by applying a solution of silver nitrate to the cut. Blood samples were obtained just before transplantation (day 0), daily for 10 days, and at days 21 and 28 after transplantation. Autopsy was performed on each animal that died and at the end of the study period at 28 days. A total of 45 Gunn rats were grouped as follows:

Group 1: IEH transplant (5 x lo7 hepatocytes) (n = 10) Group 2: FHC transplant (5 x lo7 hepatocytes) (n = 11) Group 3: Control (empty) microcapsules (5 ml) (n = 12) Group 4: Nontransplanted control (n = 12) All animals were weighed daily and received a daily diet of

standard rat chow (Ralston Purina, St. Louis, MO) and water ad libitum. Serum ALT activity (ABASOO Autoanalyzer, Abbott Laboratories, Chicago, IL) was measured in the control and treated rats.

Histology. Light and electron microscopy were performed on the transplanted IEH at the termination of the experiment at 4 wk after transplantation. The IEH were dissected from the animals and fixed in either phosphate-buffered formal- dehyde or 2% cacodylate-buffered glutaraldehyde using standard techniques. Sections for light microscopy were stained with hematoxylin and eosin. Electron microscopy sections were stained with uranyl acetate and counterstained with lead citrate.

Statistics. Student's t test, ANOVA and ANCOVA statistics were performed on the bilirubin data.

RESULTS Serum Bilirubin Levels in Gunn Rats Aper Trans-

plantation of Microencapsulated or FHC (Unencapsu- lated), Control (Empty) Microcapsules or no Microcap- sules. Figure 1 and Table 1 show the serum bilirubin data from the Gunn rats in groups 1 through 4. A highly significant (p c 0.001) decrease in serum bilirubin levels was seen in the group 1 animals that received IEH transplants. In these animals a biphasic reduction in serum bilirubin levels was observed. In the initial phase, which reached a minimum at day 4 after transplan-

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FIG. 2. One month after transplantation. Many individual IEH are seen attached throughout the omentum. Large aggregates of IEH can be seen along the porta hepatis. pancreas and the surface of the liver furrows). Extensive network of newly formed blood vessels infiltrates the clusters of IEH. No inflammatory reaction to the transplanted IEH or signs of hepatocyte rejection are visible.

FIG. 3. IEH 1 mo after transplantation are arranged in a latticelike formation. Infiltrating this lattice, newly formed blood vessels (arrows, are juxtaposed to the IEH. Surrounding each IEH is a layer of connective tissue. but no inflammatory cells are visible. The IEH appear histologically normal (original magnification x 125,.

tation, bilirubin levels fell to nearly 53% of their initial value of 7.715 ? 0.556 mg/dl. This reduction was then followed by a mild transitional rise in serum bilirubin, which peaked at day 6 after transplantation. Thereafter serum bilirubin levels fell again and were stabilized at about 35% to 45% of their pretransplantation values. This latter decrease in the bilirubin level was sustained for at least 1 mo.

Group 2, Gunn rats transplanted with free unencapsulated hepatocytes, showed only a mild lowering of serum bilirubin levels. This decrease was not statisti- cally different from pretransplantation levels.

Group 3, Gunn rats transplanted with empty (control) microcapsules, behaved as expected with no significant reduction in their serum bilirubin levels.

Group 4, controls that were not transplanted, also failed to show any significant reductions in serum bilirubin levels.

Although there appears to be some variability in the bilirubin values on day 0 (i.e., pretransplant levels) between groups 1, 2, 4 and 3, ANOVA and ANCOVA statistics revealed that the day 0 differences in serum bilirubin levels have no relationship to the serum bilirubin values on subsequent days. That is, the serum

1346 DIXIT ET AL. HE PATOLOGY

FIG. 4. Transmission electron microscopy of the intact IEH 1 mo after transplantation reveals many normal hepatocytes with well-preserved nuclei (N) and typical smooth and rough endoplasmic reticulum (ERJ. Numerous mitochondria fM), lysosomes fL) , glycogen fG) and Golgi apparatus (GA) are visible (original magnification: (a) x 11,000; tb to D x 33,000).

bilirubin levels on the days after transplantation are not dependent on day 0 values; different groups behave differently because of the type of treatment (i.e., transplantation) with a p c 0.001. Gross Morphology of the Transplanted IEH a t 4 Wk

After Transplantation. The macroscopic appearance of transplanted IEH at 4 wk after transplantation is shown in Figure 2. Numerous individual IEH were seen attached throughout the omentum, but large aggregates of IEH were attached along the portal tract, pancreas and surface of the liver. On careful examination of these regions, an extensive network of newly formed blood vessels was observed infiltrating the clusters of IEH. No inflammatory reaction to the transplanted IEH or signs of hepatocyte rejection were visible on gross examination of the peritoneal cavity.

Light Microscopy of IEH 4 Wk Afrer Transplantation. Aggregates of IEH were dissected out and were formalin fmed for light microscopy according to standard techniques. The IEH were seen arranged in a latticelike formation (Fig. 3). Infiltrating this lattice, newly formed blood vessels were juxtaposed to the microcapsules containing the hepatocytes. Surrounding each microcapsule was a layer of connective tissue, but no inflam-

matory cells were seen in or around the intact IEH in the conglomerates. The hepatocytes contained within the microcapsules appeared normal and were attached to the collagen matrix, one of the components of the microcapsule. The IEH stained positive by PAS staining, indicating the presence of glycogen in the encapsulated hepatocytes 1 mo after transplantation. Some samples of the IEH aggregates revealed many IEH in varying degrees of breakdown. In these samples an inflammatory response, possibly on the basis of rejection of the transplanted IEH, was evident. The broken microcapsules showed infiltration of inflammatory cells and giant cells engulfing the donor hepatocytes.

Electron Microscopy of the IEH 4 Wk After Transplan- tation. Transmission electron microscopy of the intact IEH revealed many normal looking hepatocytes with well-preserved nuclei and typical smooth and rough endoplasmic reticulum (Fig. 4). Numerous mitochondria, lysosomes, glycogen and Golgi apparatus were also visible throughout the encapsulated hepatocytes that had been transplanted for 1 mo in the Gunn rat. In contrast, the transplanted FHC could not be re- covered. Thus no ultrastructural studies were possible in group 2.

DISCUSSION

Microencapsulation, as demonstrated in our studies, provides a unique and effective technique for the transplantation of foreign biological substances without the need for immunosuppression. Chang and Poz- nansky (28) first demonstrated the feasibility of this approach by using microencapsulated enzymes for treating enzyme (catalase) deficiency in acatalasemic mice. With this technique microencapsulated aspara- ginase has also been successfully used for the sup- pression of asparagine-induced tumors in C3H mice ( 29). Similarly, Sun et al. (30 I have successfully treated streptozotocin-induced diabetes in rats by transplanting microencapsulated islets of Langerhans. We and others have recently demonstrated the feasibility of this approach for the replacement of liver function by transplanting IEH in the galactosamine-induced, fulminant hepatic failure rat model (22,231. However, these earlier studies did not elucidate clearly the long-term effectiveness of IEH transplantation. In the present study we have used the Gunn rat model to demonstrate the feasibility of IEH transplantation for the replacement of a specific liver function for a period of 1 mo.

Demetriou et al. (5 ) have demonstrated the long-term effectiveness of hepatocyte transplantation for the treatment of inborn errors of liver metabolism. Using serum bilirubin levels as the end point we were able to confirm the observations of Demetriou et al. that isolated hepatocytes are able to provide significant liver function to Gunn rats with a congenital enzymatic deficiency of glucuronyltransferase. In contrast to Dem- etriou et al.'s studies in which isolated hepatocytes were grown on the exterior surface of collagen-coated micro- carriers before transplantation. our technique enables hepatocytes to be microencapsulated within a three- dimensional microenvironment composed of a collagen matrix separated from the external environment by a semipermeable membrane. The semipermeable membrane allows permeant molecules, such as glucose, albumin and clotting factors, to diffuse freely across but prevents the microencapsulated materials from getting out. Recently, Goosen et al. ( 3 1 I have reported a study on the optimization of microencapsulation parameters in the alginate-polylysine microencapsulation system. It was reported and later confirmed in unpublished find- ings by our group that the permeability of the alginate- polylysine microcapsule membrane was directly propor- tional to the molecular weight of the polylysine used to prepare the microcapsule. Our unpublished permeability studies of the alginate-poly 1.-lysine membrane microcapsule showed that the microcapsules in question were permeable to molecules up to 120,000 Da. This membrane also prevented unwanted substances, such as cells and antibodies, from entering the microcapsule. Thus, by using the principle of microencapsulation, it is possible to overcome immunological tissue rejection, which hindered earlier studies in this area.

Serum bilirubin levels were significantly (p < 0.001 ) lowered by 35% to 45% in the animals receiving IEH

transplants. Free unencapsulated hepatocytes were in- effective in providing liver function to the Gunn rats. The animals transplanted with unencapsulated FHC did not differ significantly from the two control groups (groups 3 and 4). Our studies revealed an unexpected biphasic pattern of reduction in serum bilirubin levels in the IEH-transplanted animals. The first phase of the decrease occurred at 4 days after IEH transplantation, the time period at which a connective tissue capsule was seen to form around the IEH aggregates. The formation of this capsule may have caused the temporary mild rise in bilirubin levels by inhibiting diffusion across the microcapsule wall. At approximately 6 days after transplantation, we hypothesize that newly formed blood vessels infiltrated the IEH aggregates, thus facilitating the diffusion of nutrients across the microcapsule wall and giving rise to the second phase of the bilirubin decrease. This phase was sustained for approximately 4 wk. The reduction in serum bilirubin levels in our study was not as drastic as that reported by Demetriou et al. (5). This may be due to the higher viability (100%) of hepatocytes transplanted in these earlier studies. Rou- tinely, we were only able to obtain hepatocytes with a viability between 85% and 90%. By transplanting higher viability ( 100%) IEH our serum bilirubin decrease may have been proportionately more similar to that of Demetriou et al.

Although direct demonstration of bilirubin conjugates in the bile of the IEH-transplanted animals would un- equivocally demonstrate that the decrease in serum bilirubin levels was due to transplanted hepatocyte function, the primary aim of our study was to demonstrate the long-term effectiveness of IEH transplantation in Gunn rats without the need for immunosuppression. The decrease in serum bilirubin levels and subsequent increase in bilirubin conjugates in the bile of Gunn rats after hepatocyte transplantation has been previously demonstrated by several investigators (5,32 I . Recently, Demetriou et al. ( 5 ) also demonstrated this function when they transplanted microcarrier-attached hepatocytes into Gunn rats. As indicated earlier, the only difference between our hepatocyte transplantation system (IEH) and Demetriou's system (microcarrier- attached hepatocytes) is that we microencapsulated our hepatocytes before transplantation. Thus we do not expect an alternate mechanism for bilirubin conjugation to what was demonstrated by Demetriou et al. Addi- tionally, in our experimental design we included appro- priate controls to ensure that the decrease in serum bilirubin levels, after IEH transplantation, was not an artifact or a result of chance. Two different control groups were included in our study. One control group consisted of Gunn rats that were transplanted with empty microcapsules containing no hepatocytes. The other control group consisted of Gunn rats that received no transplants. In both these groups there was no significant decrease in serum bilirubin levels after transplantation. However. when compared with Gunn rats that received IEH transplantation, the IEH-transplanted

1348 DIXIT ET AL. HEPATOLOGY

Gunn rats demonstrated a significant and sustained decrease in serum bilirubin levels for up to 1 mo. Student’s t test, univariate and multivariate repeated measures statistical (ANOVNANCOVA) analyses were also con- ducted to evaluate the overall differences among the experimental groups. The ANOVNANCOVA F-statistic was greater than 15, indicating that it was highly im- probable (p c 0.001) that the difference between the IEH-transplanted and control groups was due to chance. Thus the decrease in serum bilirubin levels was most likely due to the functioning of the transplanted microencapsulated hepatocytes.

The histological evidence also correlates with the observations of Demetriou et al. in that the transplanted hepatocytes appeared histologically normal by both light and electron microscopy (15). The presence of well- preserved cellular organelles supports our view that the transplanted IEH were able to function normally to supplement deficient liver function. It should be empha- sized that our animals were not immunosuppressed and that the encapsulated hepatocytes functioned effectively without being rejected by the host animal. In those microcapsules that had broken down after transplantation, some inflammatory response, possibly on the basis of rejection of the transplanted IEH, was observed. The intact microcapsules protected the IEH from the rejection process.

Although IEH transplantation successfully reduced hyperbilirubinemia in Gunn rats for up to 1 mo, longer term (i.e., > 1 mo) efficacy of IEH transplantation needs to be assessed. Recently, some investigators have reported a progressive loss of hepatocyte function in intraperitoneally transplanted hepatocytes 2 mo after transplantation (15). If hepatocyte transplantation is limited by degenerative changes in the transplanted hepatocytes, it may be necessary to study the possibility of multiple IEH transplantation. We are currently in- vestigating this possibility.

In recent years, the development of new, more physiological attachment substrates such as EHS Gel (33) and the commercially available Matrigel (Collaborative Re- search, Boston, MA), a synthetic material that closely resembles the basement membrane composition (collagen types I, 111, IV and V; glycoproteins; proteoglycans; hyaluronic acid and lamilin) of the hepatic sinusoids has been shown to improve the biological response of cultured hepatocytes (33). By substituting these better hepatocyte attachment substrates for Vitrogen (type I collagen; Collagen Corp., Palo Alto, CAI, it may be possible to improve the functional response of transplanted IEH. Another way to improve the functional response of the transplanted IEH may be to coculture (34) the IEH before transplantation.

In this study we have shown that IEH are effective in restoring glucuronyltransferase activity in the Gunn rat. Serum bilirubin levels were significantly reduced immediately after transplantation, and transplanted IEH continued to function effectively for up to 1 mo without the need for immunosuppression. Histological evidence presented here demonstrates that the trans-

planted IEH appeared to be normal, with the typical architecture of normal hepatocytes. Our previously published studies have shown that IEH are not subject to rejection and function normally in tissue culture (22). Additionally, they were shown to provide sufficient hepatic function in conditions of acute liver failure to increase significantly the survival of experimental animals.

Thus it can be concluded that IEH are effective in significantly lowering serum bilirubin levels in the Gunn rat, and transplantation of IEH is a unique approach toward the restoration of at least one liver function in congenital enzymatic liver deficiency. In the future this technique may provide a therapeutic option for the management of congenital enzyme deficiency.

Acknowledgment: We gratefully acknowledge the assistance in the ANOVA and ANCOVA statistical analysis of the data by Bradford Arthur, Ph.D., Cali- fornia State University, San Francisco.

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Restoration of liver function in gunn rats without immunosuppression using transplanted...

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